The following is a discussion of the relevant art, none of which is admitted to be prior art to the appended claims.
DNA polymerase errors occurring during PCR amplification result in the presence of mutations in the amplified product. This problem can be particularly acute with Taq DNA polymerase which lacks a proofreading exonuclease and has a base substitution error rate on the order of 1/10.sup.4 to 1/10.sup.5 nucleotides polymerized under PCR conditions (Eckert and Kunkel, Nucleic Acids Res., 18:3739, 1990; Mattila et al.,Nucleic Acids Res., 19:4967, 1991; Saiki et al., Science, 239:487, 1988; Tindall and Kunkel, Biochemistry, 27:6008, 1988). The significance of error rates of this magnitude has been pointed out by Keohavong and Thilly (Keohavong and Thilly, Proc. Natl. Acad. Sci. U.S.A., 86:9253, 1989), who noted that at a misincorporation rate of 2/10.sup.4, 10.sup.6 -fold (twenty cycle) amplification of a 100 base pair sequence yields a population of product molecules, each of which has an 80% probability of containing a mutation somewhere in its sequence (Keohavong and Thilly, supra). The frequency of polymerase errors during PCR can be estimated from equations 1 and 6 of Luria and Delbruck (Luria, S. E. & Delbruck, M. Genetics 28:491-511, 1943) as EQU f=2lNa (Equation 1)
where f is the expected fraction of product molecules that contain a mutation somewhere in their sequence, l is the length of the amplified segment in bp, N is the number of cycles, and a is the error rate for the polymerase expressed per nucleotide incorporated. This problem has been alleviated to some extent by identification of the thermostable Pfu and Tli (Vent.TM.) DNA polymerases, which have proofreading activity and display a two to ten-fold improvement in fidelity relative to Taq (Lundberg et al., Gene, 108:1, 1991; Mattila et al., supra). However, given that the probability of polymerase misincorporation event per cycle is also proportional to the size of the sequence being amplified, polymerase-generated mutations remain a significant problem for extensive amplification of sequences in the kilobase range.